Talk and Poster: Gordon Research Conferences : Mitochondria and Chloroplasts, Plymouth , NH (1996) To determine if the Green Fluorescent Protein (GFP) can be used as a sub-cellular marker in plant cells in vivo we transformed tobacco suspension cells and tobacco and Petunia plants with a constitutively expressed GFP with and without a mitochondrial localization signal. Green fluorescence is visible by eye when calli expressing GFP or targeted GFP are exposed to UV light. Confocal or two photon laser scanning microscopy reveals a distinct sub-cellular localization of fluorescence in cells expressing GFP or targeted GFP. In NTI cells expressing GFP, fluorescence is distributed throughout the cytoplasm but seems to be excluded from compartments that are surrounded by selective membranes. GFP is not visible in vacuoles or nucleoli and is excluded from round spaces in the cytoplasm that seem to be organelles such as leucoplasts or amyloplasts. In contrast, in cells expressing the mitochondrially-targeted GFP fusion protein, fluorescence is restricted to particles of about 1 to 2 mm in diameter that are visible only in the cytoplasm, but not in vacuoles or the nucleoplasm. The mitochondrially targeted GFP fusion protein co-localizes with the mitochondria-specific dye MitoTrackerTM CMTM ROS-H2 showing that the coxIVGFP is targeted specifically to mitochondria. Preliminary results from green plants indicate that GFP localized to mitochondria permits the direct observation of mitochondria in vivo throughout the life of a green plant. This gives new insight into developmental and tissue specific changes in shapes and movements of mitochondria. Three-dimensional reconstruction can provide additional information about the distribution and number of mitochondria in living cells and tissues.